Characterization of microchannel plate detector response for the detection of native multiply charged high mass single ions in orthogonal‐time‐of‐flight mass spectrometry using a Timepix detector

Abstract Time‐of‐flight (TOF) systems are one of the most widely used mass analyzers in native mass spectrometry (nMS) for the analysis of non‐covalent multiply charged bio‐macromolecular assemblies (MMAs). Typically, microchannel plates (MCPs) are employed for high mass native ion detection in TOF MS. MCPs are well known for their reduced detection efficiency when impinged by large slow moving ions. Here, a position‐ and time‐sensitive Timepix (TPX) detector has been added to the back of a dual MCP stack to study the key factors that affect MCP performance for MMA ions generated by nMS. The footprint size of the secondary electron cloud generated by the MCP on the TPX for each individual ion event is analyzed as a measure of MCP performance at each mass‐to‐charge (m/z) value and resulted in a Poisson distribution. This allowed us to investigate the dependency of ion mass, ion charge, ion velocity, acceleration voltage, and MCP bias voltage on MCP response in the high mass low velocity regime. The study of measurement ranges; ion mass = 195 to 802,000 Da, ion velocity = 8.4 to 67.4 km/s, and ion charge = 1+ to 72+, extended the previously examined mass range and characterized MCP performance for multiply charged species. We derived a MCP performance equation based on two independent ion properties, ion mass and charge, from these results, which enables rapid MCP tuning for single MMA ion detection.

(MS) instrumentation [10][11][12] further extended the mass range to several megadaltons, by enabling the ionization and analysis of noncovalent bio-macromolecular assemblies (MMAs) in their pseudonative state, [13][14][15] where quaternary structure is retained, a method referred to as native MS (nMS). 16 Time-of-flight (TOF) MS is one of the most commonly used mass analyzers for high mass detection due to its unlimited theoretical mass range, high sensitivity, and speed of analysis. 17,18 Ion detection in TOF MS is traditionally accomplished using microchannel plates (MCPs) because of their high gain, fast response, and large active area. [19][20][21] However, MCP detectors suffer from a reduced detection efficiency when impinged by large slowly moving ions. [22][23][24][25][26] Hence, the key parameters affecting the detection of high mass non-covalent ions generated by nMS must be better understood. Here, we conducted a detailed study to gain insight into the influence of critical ion and ion optical parameters on MCP detector performance for high mass multiply charged slow moving protein/protein complex ions, using the spatially and temporally resolved detection of individual ion events.
In previous studies, the performance of the MCP has been examined for the detection of singly charged biomolecules of mass up to 290 kDa, by measuring the secondary electron yield (γ, average number of electrons produced within the MCP per initial ion impact event) and/or detection efficiency (ε, probability of generation of one or more secondary electrons during the initial ion impact event), by comparing the ion counts at different acceleration voltages, 22 using a superconducting tunnel junction (STJ) detector 24 or an inductive charge detector (ICD) 25,26 in parallel with MCP detector. Secondary electron emission from the impacts of low velocity macro-ions of m/ z < 100,000 Da from various conducting surfaces has also been studied. [27][28][29][30][31] In this work, we have added an active pixelated detector (Timepix [TPX]) to the back of a dual MCP stack on a modified orthogonal reflectron TOF (O/R-TOF) MS (LCT) equipped with a nanoESI source. 32 With this unique setup, we analyzed the individual footprints of secondary electron clouds generated by the MCP on the TPX detector. As the size of each electron cloud depends on the number of electrons produced from an individual ion impact due to its space-charge-driven expansion, this allowed us the study of MCP amplification as a function various parameters for multiply charged MMA ions of molecular weights up to 802 kDa.
The TPX is a position-and time-sensitive charge detector consisting of a 512 Â 512 pixel array with each pixel capable of recording both the arrival time and impact coordinates of impinging particles. 33 Despite the fact that TPX technology has its origin in high energy physics, 34-37 the integration of TPX with MCP amplifier allowed the detection of low energy particles, [38][39][40][41] which extended its scope to MS applications. 21 MCP-TPX assembles have been used in MS with the goals (i) to improve the spatial resolution and throughput of MS imaging [42][43][44][45] ; (ii) to investigate the ion transport properties through different ion optical elements of MS 32,46,47 ; and (iii) for the enhanced detection of high mass ions. 32,48,49 A previous study from our group conducted on the same TPX equipped LCT system has demonstrated the capability of TPX to detect non-covalent protein complexes and to image single ion events. 32 We present a detailed characterization of MCP response for high mass multiple charged non-covalent species using the single ion imaging capability of TPX detector. We analyzed the electron cloud footprints on the TPX correspond to each mass-to-charge (m/z) value from a set of 16 samples that encompasses the following measure-

| Mass spectrometer and detection system
All experiments were performed on a modified LCT nESI-O-TOF mass spectrometer (Micromass, Manchester, UK) equipped with a TPX detector that has recently been described in detail in reference. 32 Samples were introduced into the mass spectrometer using homemade gold-coated needles via an in-house built static nanoESI source.
Ions are transferred into an orthogonal acceleration TOF mass analyzer for TOF separation via two differentially pumped hexapole RF lenses. The instrument has been modified for improved transmission of high m/z ions as described previously. 32 The detector assembly consist of an MCP All samples were characterized using a Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) prior to the measurements on the LCT-TPX system for the TOF to m/z conversion.

| Data acquisition and analysis
The SoPhy (Software for Physics) software package Version 1.5.2 was used for TPX chip control and data acquisition (Amsterdam Scientific Instruments, Amsterdam, The Netherlands). A total of 5000-10,000 TOF cycles (frames) were collected and summed for each dataset.
SoPhy generates a binary 512 Â 512 frame for each TOF cycle. Every x-y position (pixel) in the frame contains information on the number of clock counts passed since the start of the TOF cycle and the arrival of sufficient charge to trigger that pixel. One hundred frames are bundled in a zipped output file. An in-house developed algorithm written in MATLAB (R2014a, MathWorks Inc., Natick, MA, USA) was used to convert the data to NetCDF format and to select sub-frames from frames and TOF ranges. The MATLAB functions "bwlabel" and "regionprops" were used to detect and measure the properties of connected areas (pixel clusters) in 2D binary sub-frames. The secondary electron multiplicity is described by a Poisson distribution given by where P n is the probability of emitting n electrons from the MCP and γ is the average number of electrons emitted per initial ion impact, known as secondary electron yield. 25 The detection efficiency, ε, is defined as the probability of emitting at least one electron from a single ion impact and given by where P 0 is the probability of not emitting any electrons and calcu- ions impinge the MCP detector with a lower velocity, resulting in reduced γ, making it harder to detect large slow moving ions. [22][23][24][25][26] Several groups previously examined the performance of the MCP in high mass low velocity regime by measuring γ and/or ε with different approaches. Macfarlane's group has calculated ε through the following procedure. First, the ion intensity at a high acceleration potential was measured to determine the integrated intensity of particular ions under conditions where P(γ) = 1 (i.e., γ is a large number).
Then, the integrated intensity of the same ions was measured at the velocity of interest by reducing the acceleration voltage. Later, the ratio of two intensities was taken for the calculation of ε. Note that the ion intensity was derived using the single ion counting technique.
The study was conducted for the following measurement range: mass = 86-5734 Da, charge = 1, velocity = 13-32 km/s, and acceleration voltage ≤ 20 kV. 22 Benner's group measured ion intensity using MCP along with STJ detector, which has 100% detection efficiency, The detection in TPX is frame based, and each frame corresponds to a single TOF cycle. Figure 1A

| Influence of ion properties on MCP performance
In this section, single ion imaging capability of TPX has been utilized to study the influence of ion properties on ion to electron conversion factor and thus the MCP response. γ is generally expressed as a function of two dependent parameters, ion mass and velocity, [24][25][26] given where the k 0 is constant of proportionality, and a and b are fit parame-

ters. Combining Equation (3) with basic TOF MS equation
yields the relation Equation (5) can be rewritten as a function of two independent parameters, ion mass and charge, at a given acceleration voltage as where In this study, instead of calculating γ, we have measured the mean n p (μ) that corresponds to each m/z or TOA, which is proportional to γ.  Figure S2 shows the TOF to m/z conversion curve, plotted by comparing LCT measured TOF data with the Orbitrap m/z spectrum of each sample. From Equation (4) and calibration curve, v can be written as k 2 TOF À0.99 , where k 2 = (1.142 Â 10 6 )k 0 . Therefore, Equations (3) and 6 can rewritten to calculate μ as where k 3 = (μ/γ)k 1 and k 4 = (μ/γ)k 2 .

| Influence of ion optics on MCP performance
We next investigated the influence of ion optics on the MCP detection efficiency. In our previous work, we have shown that the voltage settings "ion energy" and "RF DC offset voltage of first hexapole" have an influence on the axial ion energy of the ion beam, while "TOF tube voltage" affects the orthogonal ion energy. 32 An increase in ion to electron conversion efficiency has been expected with a raise in orthogonal voltage as per Equation (5). Figure 5A shows the increasing trend of μ with the orthogonal TOF tube voltage. As per Equation (5) Figure 4D. Therefore, it is difficult to fit a power function to TOF tube voltage-μ curve with the limited number of data points. As expected, μ value remains unaffected by voltage settings "RF DC offset of first hexapole" and "ion energy," because both these voltages contribute to the total energy of the ion beam only through the axial velocity component, which is parallel to the detection plane.
As discussed in the previous section, a higher MCP bias voltage increases the number of secondary electrons generated in each amplification step within the MCP and thus raises μ ( Figure 5B). V TPX-MCP back , the potential gradient between MCP back plate and TPX detector, is one of the parameters that has an influence on the overall detector performance. Figure 5C shows the dependence of μ on the V TPX-MCP back . At a lower V TPX-MCP back , the potential gradient is not strong enough to accelerate and focus the electron clouds from the MCP to the TPX at the low voltage. Most importantly, the charge deposited into a single pixel by the electron clouds is not adequate for the activation of TPX pixels. A higher amount of charge is deposited with an increase in TPX voltage, which leads to an increase in μ. However, after attaining a maximum value, μ tends to decrease due to the space charge effect. At a higher V TPX-MCP back , electrons are accelerated much faster towards the TPX detector, providing less time for the electron cloud to expand, which leads to the activation of less number of TPX pixels. The space-charge-driven expanded shape of the electron cloud was expected to fit a cosine distribution. 50,51 Understanding the influence of ion optics on MCP detector effi-